Whole Cell Patch Clamp of Dispersed Human Islet Cells
Xiaoqing Dai, Austin Bautista, Patrick E Macdonald
Abstract
Cells use exocytosis to secrete a wide variety of molecules, including proteins, hormones, and neurotransmitters. Exocytosis can be monitored at the single-cell level by using patch-clamp electrophysiology to measure changes in membrane capacitance as vesicles fuse with the cell membrane and release their content. Dispersion of pancreatic islets into single cells allows for individual characterization of electrophysiological characteristics and allows for collection of cellular content for recovery of full-length transcriptomes by use of Smart-seq2.
Described in this protocol is the dispersion of pancreatic islets into single cells followed by whole-cell patch clamp electrophysiology which includes parameters representing cell size, exocytosis, sodium channel currents, and calcium channel currents. Cells are then collected individually after recording to be processed for single-cell RNA sequencing.
Steps
Procedure
On the day receiving the shipped human pancreatic islets, hand-picked islets are dissociated to single cells using StemPro accutase (Gibco/Fisher, A11105-01). Plate cells in 35 mm cell culture dishes, and culture in DMEM with 5.5 mM glucose, 10% FBS, and 100 U/mL penicillin/ streptomycin for 1-4 days.
Start patch clamping single cells after one overnight incubation, and continue patching cells for up to 4 days. Electrical activities are measured by using a pipette coated with sylgard (3 ~ 5 MΩ) in a heated chamber (32–35°C). Quality control is assessed by the stability of seal (>10 GΩ) and access resistance (<15 MΩ).
Perform electrical activity measurements in 1 minute from “break in” of cell membrane; measurement protocols include (in order): exocytosis, voltage-gated Na and Ca channel currents activated at -10 mV, and -120 mV, voltage-gated Na and Ca channel currents activated from -60 to +30 mV, steady-state inactivation of voltage-gated Na channel currents, reversal potential, hyperpolarization-activated non-selective cation currents activated at -140 mV.
When finishing all the measurements, use another big-tip pipette (0.2 ~ 0.5 MΩ) prefilled with 0.5mL lysis mix, suck the cell into the pipette, and transfer it into a 0.2mL PCR tube prefilled with 4mL lysis mix.
Save cells in -80°C freezer before shipping out for sequencing.
Data Analysis
Using the software of Fitmaster (HEKA Instruments Inc, Germany), analysis is performed on the level of recording traces.
